Ceramides improve cardiovascular risk prediction beyond low-density lipoprotein cholesterol

Aims Low-density lipoprotein cholesterol (LDL-C) is the best documented cardiovascular risk predictor and at the same time serves as a target for lipid-lowering therapy. However, the power of LDL-C to predict risk is biased by advanced age, comorbidities, and medical treatment, all known to impact cholesterol levels. Consequently, such biased patient cohorts often feature a U-shaped or inverse association between LDL-C and cardiovascular or overall mortality. It is not clear whether these constraints for risk prediction may likewise apply to other lipid risk markers in particular to ceramides and phosphatidylcholines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods In this observational cohort study, we recorded cardiovascular mortality in 1195 patients over a period of up to 16 years, and results comprising a total of 12 262 patient-years. The median age of patients at baseline was 67 years. All participants were either consecutively referred to elective coronary angiography or diagnosed with peripheral artery disease, indicating a high cardiovascular risk. At baseline, 51% of the patients were under statin therapy. We found a U-shaped association between LDL-C and cardiovascular mortality with a trough level of around 150 mg/dL of LDL-C. Cox regression analyses revealed that LDL-C and other cholesterol species failed to predict cardiovascular risk. In contrast, no U-shaped but linear association was found for ceramide- and phosphatidylcholine-containing markers and these markers were able to significantly predict the cardiovascular risk even after multivariate adjustment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion We thus suggest that ceramides- and phosphatidylcholine-based predictors rather than LDL-C may be used for a more accurate cardiovascular risk prediction in high-risk patients.Peer reviewe

Identification of Hypoxia-Induced Genes in Human SGBS Adipocytes by Microarray Analysis

Hypoxia in adipose tissue is suggested to be involved in the development of a chronic mild inflammation, which in obesity can further lead to insulin resistance. The effect of hypoxia on gene expression in adipocytes appears to play a central role in this inflammatory response observed in obesity. However, the global impact of hypoxia on transcriptional changes in human adipocytes is unclear. Therefore, we compared gene expression profiles of human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes under normoxic or hypoxic conditions to detect hypoxia-responsive genes in adipocytes by using whole human genome microarrays. Microarray analysis showed more than 500 significantly differentially regulated mRNAs after incubation of the cells under low oxygen levels. To gain further insight into the biological processes, hypoxia-regulated genes after 16 hours of hypoxia were classified according to their function. We identified an enrichment of genes involved in important biological processes such as glycolysis, response to hypoxia, regulation of cellular component movement, response to nutrient levels, regulation of cell migration, and transcription regulator activity. Real-time PCR confirmed eight genes to be consistently upregulated in response to 3, 6 and 16 hours of hypoxia. For adipocytes the hypoxia-induced regulation of these genes is shown here for the first time. Moreover in six of these eight genes we identified HIF response elements in the proximal promoters, specific for the HIF transcription factor family members HIF1A and HIF2A. In the present study, we demonstrated that hypoxia has an extensive effect on gene expression of SGBS adipocytes. In addition, the identified hypoxia-regulated genes are likely involved in the regulation of obesity, the incidence of type 2 diabetes, and the metabolic syndrome

Coronary Event Risk Test (CERT) as a Risk Predictor for the 10-Year Clinical Outcome of Patients with Peripheral Artery Disease

(1) Background: Ceramides are a new kind of lipid biomarker and have already been demonstrated to be valuable risk predictors in coronary patients. Patients with peripheral artery disease (PAD) are a population with a worse prognosis and higher mortality risk compared to coronary artery disease (CAD) patients. However, the value of ceramides for risk prediction in PAD patients is still vague, as addressed in the present study. (2)Methods: This observational study included 379 PAD patients. The primary endpoint was all-cause mortality at 10 years of follow-up. A set of ceramides was measured by LC-MS/MS and combined according to the Coronary Event Risk Test (CERT) score, which categorizes patients into one of four risk groups (low risk, moderate risk, high risk, very high risk). (3) Results: Kaplan–Meier survival curves revealed that the overall survival of patients decreased with the increasing risk predicted by the four CERT categories, advancing from low risk to very high risk. Cox regression analysis demonstrated that each one-category increase resulted in a 35% rise in overall mortality risk (HR = 1.35 [1.16–1.58]). Multivariable adjustment, including, among others, age, LDL-cholesterol, type 2 diabetes, and statin treatment before the baseline, did not abrogate this significant association (HR = 1.22 [1.04–1.43]). Moreover, we found that the beneficial effect of statin treatment is significantly stronger in patients with a higher risk, according to CERT. (4) Conclusions: We conclude that the ceramide-based risk score CERT is a strong predictor of the 10-year mortality risk in patients with PAD.Peer reviewe

Relations between lipoprotein(a) concentrations, LPA genetic variants, and the risk of mortality in patients with established coronary heart disease: a molecular and genetic association study

Background: Lipoprotein(a) concentrations in plasma are associated with cardiovascular risk in the general population. Whether lipoprotein(a) concentrations or LPA genetic variants predict long-term mortality in patients with established coronary heart disease remains less clear. Methods: We obtained data from 3313 patients with established coronary heart disease in the Ludwigshafen Risk and Cardiovascular Health (LURIC) study. We tested associations of tertiles of lipoprotein(a) concentration in plasma and two LPA single-nucleotide polymorphisms ([SNPs] rs10455872 and rs3798220) with all-cause mortality and cardiovascular mortality by Cox regression analysis and with severity of disease by generalised linear modelling, with and without adjustment for age, sex, diabetes diagnosis, systolic blood pressure, BMI, smoking status, estimated glomerular filtration rate, LDL-cholesterol concentration, and use of lipid-lowering therapy. Results for plasma lipoprotein(a) concentrations were validated in five independent studies involving 10 195 patients with established coronary heart disease. Results for genetic associations were replicated through large-scale collaborative analysis in the GENIUS-CHD consortium, comprising 106 353 patients with established coronary heart disease and 19 332 deaths in 22 studies or cohorts. Findings: The median follow-up was 9·9 years. Increased severity of coronary heart disease was associated with lipoprotein(a) concentrations in plasma in the highest tertile (adjusted hazard radio [HR] 1·44, 95% CI 1·14–1·83) and the presence of either LPA SNP (1·88, 1·40–2·53). No associations were found in LURIC with all-cause mortality (highest tertile of lipoprotein(a) concentration in plasma 0·95, 0·81–1·11 and either LPA SNP 1·10, 0·92–1·31) or cardiovascular mortality (0·99, 0·81–1·2 and 1·13, 0·90–1·40, respectively) or in the validation studies. Interpretation: In patients with prevalent coronary heart disease, lipoprotein(a) concentrations and genetic variants showed no associations with mortality. We conclude that these variables are not useful risk factors to measure to predict progression to death after coronary heart disease is established. Funding: Seventh Framework Programme for Research and Technical Development (AtheroRemo and RiskyCAD), INTERREG IV Oberrhein Programme, Deutsche Nierenstiftung, Else-Kroener Fresenius Foundation, Deutsche Stiftung für Herzforschung, Deutsche Forschungsgemeinschaft, Saarland University, German Federal Ministry of Education and Research, Willy Robert Pitzer Foundation, and Waldburg-Zeil Clinics Isny

Associations of Polymorphisms in the Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1 Alpha Gene With Subsequent Coronary Heart Disease : An Individual-Level Meta-Analysis

Background: The knowledge of factors influencing disease progression in patients with established coronary heart disease (CHD) is still relatively limited. One potential pathway is related to peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PPARGC1A), a transcription factor linked to energy metabolism which may play a role in the heart function. Thus, its associations with subsequent CHD events remain unclear. We aimed to investigate the effect of three different SNPs in the PPARGC1A gene on the risk of subsequent CHD in a population with established CHD.Methods: We employed an individual-level meta-analysis using 23 studies from the GENetIcs of sUbSequent Coronary Heart Disease (GENIUS-CHD) consortium, which included participants (n = 80,900) with either acute coronary syndrome, stable CHD, or a mixture of both at baseline. Three variants in the PPARGC1A gene (rs8192678, G482S; rs7672915, intron 2; and rs3755863, T528T) were tested for their associations with subsequent events during the follow-up using a Cox proportional hazards model adjusted for age and sex. The primary outcome was subsequent CHD death or myocardial infarction (CHD death/myocardial infarction). Stratified analyses of the participant or study characteristics as well as additional analyses for secondary outcomes of specific cardiovascular disease diagnoses and all-cause death were also performed.Results: Meta-analysis revealed no significant association between any of the three variants in the PPARGC1A gene and the primary outcome of CHD death/myocardial infarction among those with established CHD at baseline: rs8192678, hazard ratio (HR): 1.01, 95% confidence interval (CI) 0.98-1.05 and rs7672915, HR: 0.97, 95% CI 0.94-1.00; rs3755863, HR: 1.02, 95% CI 0.99-1.06. Similarly, no significant associations were observed for any of the secondary outcomes. The results from stratified analyses showed null results, except for significant inverse associations between rs7672915 (intron 2) and the primary outcome among 1) individuals aged >= 65, 2) individuals with renal impairment, and 3) antiplatelet users.Conclusion: We found no clear associations between polymorphisms in the PPARGC1A gene and subsequent CHD events in patients with established CHD at baseline.Peer reviewe

Subsequent Event Risk in Individuals with Established Coronary Heart Disease:Design and Rationale of the GENIUS-CHD Consortium

BACKGROUND: The "GENetIcs of sUbSequent Coronary Heart Disease" (GENIUS-CHD) consortium was established to facilitate discovery and validation of genetic variants and biomarkers for risk of subsequent CHD events, in individuals with established CHD. METHODS: The consortium currently includes 57 studies from 18 countries, recruiting 185,614 participants with either acute coronary syndrome, stable CHD or a mixture of both at baseline. All studies collected biological samples and followed-up study participants prospectively for subsequent events. RESULTS: Enrollment into the individual studies took place between 1985 to present day with duration of follow up ranging from 9 months to 15 years. Within each study, participants with CHD are predominantly of self-reported European descent (38%-100%), mostly male (44%-91%) with mean ages at recruitment ranging from 40 to 75 years. Initial feasibility analyses, using a federated analysis approach, yielded expected associations between age (HR 1.15 95% CI 1.14-1.16) per 5-year increase, male sex (HR 1.17, 95% CI 1.13-1.21) and smoking (HR 1.43, 95% CI 1.35-1.51) with risk of subsequent CHD death or myocardial infarction, and differing associations with other individual and composite cardiovascular endpoints. CONCLUSIONS: GENIUS-CHD is a global collaboration seeking to elucidate genetic and non-genetic determinants of subsequent event risk in individuals with established CHD, in order to improve residual risk prediction and identify novel drug targets for secondary prevention. Initial analyses demonstrate the feasibility and reliability of a federated analysis approach. The consortium now plans to initiate and test novel hypotheses as well as supporting replication and validation analyses for other investigators

Quercetin Impacts Expression of Metabolism- and Obesity-Associated Genes in SGBS Adipocytes

Obesity is characterized by the rapid expansion of visceral adipose tissue, resulting in a hypoxic environment in adipose tissue which leads to a profound change of gene expression in adipocytes. As a consequence, there is a dysregulation of metabolism and adipokine secretion in adipose tissue leading to the development of systemic inflammation and finally resulting in the onset of metabolic diseases. The flavonoid quercetin as well as other secondary plant metabolites also referred to as phytochemicals have anti-oxidant, anti-inflammatory, and anti-diabetic effects known to be protective in view of obesity-related-diseases. Nevertheless, its underlying molecular mechanism is still obscure and thus the focus of this study was to explore the influence of quercetin on human SGBS (Simpson Golabi Behmel Syndrome) adipocytes’ gene expression. We revealed for the first time that quercetin significantly changed expression of adipokine (Angptl4, adipsin, irisin and PAI-1) and glycolysis-involved (ENO2, PFKP and PFKFB4) genes, and that this effect not only antagonized but in part even overcompensated the effect mediated by hypoxia in adipocytes. Thus, these results are explained by the recently proposed hypothesis that the protective effect of quercetin is not solely due to its free radical-scavenging activity but also to a direct effect on mitochondrial processes, and they demonstrate that quercetin might have the potential to counteract the development of obesity-associated complications

Die Rolle von p6* für den HI-viralen Replikationszyklus

Auf Grund der Genomorganisation von HIV-1 ist das transframe-Protein p6* Bestandteil des Gag-Pol-Polyproteinvorläufers und liegt dort zwischen dem vom gag-Gen kodierten Nukleocapsid-Protein (NC) und der vom pol-Gen kodierten Protease (PR). P6* selbst wird vom 5´Ende des pol-Gens kodiert. Die Translation zweier Polyproteinvorläufer, Gag und Gag-Pol, wird durch einen -1 ribosomalen Leserastersprung an einer frame shift-Region der RNA ausgelöst. Diese hoch konservierte Region liegt im Kodierungsbereich für den Aminoterminus von p6*. Darüber hinaus ist p6* und die ebenfalls pol-kodierte PR mit p1 und p6gag des gag-Leserasters überlagert. Abgesehen von seinem Aminoterminus ist auch der Carboxylterminus von p6* hoch konserviert. In unserer Arbeitsgruppe konnte bereits gezeigt werden, dass rekombinant hergestelltes p6* die PR konzentrationsabhängig hemmt. Für diesen Effekt ist hauptsächlich das carboxylterminale Tetrapeptid von p6* verantwortlich. Darüber hinaus stellt es den p6*-Anteil der p6*-PR-Spaltstelle dar, deren Prozessierung für die Freisetzung und vollständige Aktivierung der PR essenziell ist. Mutationen am p6*-Carboxylterminus führten entweder zu einer Verzögerung oder zu einer Blockade der Prozessierung, die im Provirus HX10 unterschiedlich schwere Prozessierungsdefekte hervorriefen. Jedoch konnte für Mutationen, die die Prozessierung verzögerten, bisher kein messbarer Einfluss auf die virale Replikation belegt werden. Im Rahmen der vorliegenden Arbeit wurde in einem sensitiveren Testsystem gezeigt, dass auch geringere Prozessierungsdefekte für die entsprechenden Virusmutanten zu einem Replikationsnachteil gegenüber dem Wildtyp (Wt) führen. Um die Rolle der zentralen p6*-Region im Lebenszyklus von HIV-1 in Zusammenhang mit einer postulierten Interaktion von p6* und Nef während der Virusreplikation aufzuklären, wurden in dieser Arbeit sequentielle Mutationen in die kodierende Region von p6* eingeführt. Die Mutationen in der zentralen p6*-Region wählte man so, dass sie die überlagerten p1- und p6gag-Leserahmen unberührt ließen. Als Basis der Mutagenesestudien diente dabei das Nef-kodierende HIV-1-Isolat NL4-3. Es stellte sich heraus, dass die zentrale p6*-Region weder Einfluss auf den Leserastersprung noch auf die Produktion der Polyproteinvorläufer hat. Auch für die Aktivität der PR oder für die Inkorporation von Nef in Viruspartikel spielt sie keine Rolle. Eine Interaktion mit Nef konnte für diesen Bereich von p6* ausgeschlossen werden. Jedoch bewirkte eine Substitution der gesamten zentralen p6*-Region einen Rückgang der viralen Infektiosität und Replikation in vitro. Mutationen im p6*-Aminoterminus beeinflussen unausweichlich die frame shift-Region oder den überlagerten gag-Leserahmen. Da eine spezifische Mutagenese des p6*-Aminoterminus nicht möglich ist, ohne dabei überlagerte Strukturen zu manipulieren, ist die Rolle dieser Region für die Virusreplikation von HIV-1 noch unbekannt. Daher wurde in dieser Arbeit ein neues HI-Provirus konstruiert, bei dem die frame shift-Region so versetzt wurde, dass die beiden Leserahmen gag und pol entkoppelt vorliegen. Für dieses Virus konnte gezeigt werden, dass die neu generierte frame shift-Region einen funktionellen Leserastersprung auslöst, der zur Translation der Gag und Gag-Pol-Polyproteinvorläufer führt. Deren Prozessierung sowie Infektiosität und Replikationsfähigkeit entsprachen annähernd dem des NL4-3-Isolats. Damit repräsentiert dieses Viruskonstrukt ein neues wertvolles Werkzeug für die Analyse der HIV-1 Replikation. Es erlaubt erstmalig die frame shift-Region p1 und p6gag, sowie p6* und die PR unabhängig voneinander auch durch Deletions- oder Insertions-Mutagenese zu manipulieren. Mutationsanalysen, mit diesem neuen Virus ergaben, dass auch der p6*-Aminoterminus für die in vitro -Replikation von HIV-1 nicht essenziell ist. Auch die Deletion der gesamten zentralen p6*-Region brachte in vitro keine erkennbaren Nachteile für den Replikationszyklus mit sich. Im Gegenteil, die Virusreplikation wurde sogar beschleunigt. Weiterhin wurde festgestellt, dass Reportergene in den p6*-Leserahmen eingebracht werden können, die die zentrale p6* Region ersetzen. Mit zunehmender Größe schränken diese Insertionen die Funktionalität des Virus jedoch ein. Daher wird vermutet, dass die Aktivität der PR vom Abstand zu ihren aminoterminal gelegenen Spaltstellen im Gag-Pol-Vorläufer abhängt und dass diese Insertionen in den p6*-Leserahmen die Dimerisierung und die Faltung der PR im Gag-Pol-Vorläuferprotein stören. Diese Arbeit kommt somit zu dem Ergebnis, dass der Carboxylterminus von p6* für die PR-Aktivität und damit für die Replikation des HI-Virus von entscheidender Bedeutung ist. Im Gegensatz dazu ist die Gestalt des Aminoterminus für die Funktionalität des Virus ebenso unerheblich wie die zentrale Region von p6*. Es wird daher angenommen, dass sie die Funktion eines spacers einnimmt, um in vivo dem Virus Raum für escape-Mutationen zu bieten

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication▿

Apart from its regulatory role in protease (PR) activation, little is known about the function of the human immunodeficiency virus type 1 transframe protein p6* in the virus life cycle. p6* is located between the nucleocapsid and PR domains in the Gag-Pol polyprotein precursor and is cleaved by PR during viral maturation. We have recently reported that the central region of p6* can be extensively mutated without abolishing viral infectivity and replication in vitro. However, mutagenesis of the entire p6*-coding sequence in the proviral context is not feasible without affecting the superimposed frameshift signal or the overlapping p1-p6gag sequences. To overcome these limitations, we created a novel NL4-3-derived provirus by displacing the original frameshift signal to the 3′ end of the gag gene, thereby uncoupling the p6* gene sequence from the p1-p6gag reading frame. The resulting virus (AL) proved to be replication competent in different cell cultures and thus represents an elegant tool for detailed analysis of p6* function. Hence, extensive deletions or substitutions were introduced into the p6* gene sequence of the AL provirus, and effects on particle release, protein processing, and viral infectivity were evaluated. Interestingly, neither the deletion of 63% of all p6* residues nor the partial substitution by a heterologous sequence affected virus growth and infectivity, suggesting that p6* is widely dispensable for viral in vitro replication. However, the insertion of a larger reporter sequence interfered with virus production and maturation, implying that the length or conformation of this spacer region might be critical for p6* function

Evaluation of the association between circulating microRNAs and kidney function in coronary angiography patients.

Circulating microRNAs (miRNAs) have been linked to chronic kidney disease. Little is known about the association between circulating miRNAs and kidney function in patients at high cardiovascular risk. We therefore investigated the association between a panel of candidate miRNAs and kidney function, based on estimated glomerular filtration rate (eGFR), in two independent cohorts of patients undergoing coronary angiography. The present study totally included 438 coronary angiography patients, who were divided into a discovery cohort (n=120) and a validation cohort (n=318). A candidate miRNA panel comprising 50 renal miRNAs were selected from the literature and expression levels of circulating miRNAs were determined by real-time PCR. Out of initially tested candidate-miRNAs, 38 were sufficiently detectable in plasma. Their association with kidney function was evaluated in the discovery cohort. Associations of seven out of these miRNAs with eGFR were significant after multiple testing correction via false discovery rate (FDR) estimation. To verify obtained results, miRNAs with significant FDR were further analysed in the validation cohort. MiRNAs miR-106b-5p, miR-16-5p, miR-19b-3p, miR-20a-5p, miR-25-3p, and miR-451a proved to be significantly associated with eGFR also in the validation cohort (all p-values <0.001). Association between identified renal miRNAs and kidney function was confirmed by ANCOVA adjusting for age, gender, type 2 diabetes, hypertension, and albumin-to-creatinine ratio. In conclusion, our study showed that miR-16-5p, miR-19b-3p, miR-20a-5p, miR-25-3p, miR-106b-5p, and miR-451a are significantly linked to kidney function in coronary angiography patients